Use of the dextrogyral enantiomer of milnacipran for the preparation of a drug

ABSTRACT

The present invention concerns the use of a mixture of enantiomers enriched in the dextrogyral enantiomer of milnacipran and/or of at least one of its metabolites, as well as their pharmaceutically-acceptable salts, for the preparation of a drug intended to prevent or to treat disorders that can be managed by double inhibition of serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting the risks of cardiovascular disturbances and/or organ and/or tissue toxicity.

The present invention concerns the use of a mixture of enantiomersenriched in the dextrogyral enantiomer of milnacipran and/or of at leastone its metabolites, as well as their pharmaceutically-acceptable salts,for the preparation of a drug intended to prevent or to treat disordersthat can be managed by double inhibition of serotonin (5-HT) andnorepinephrine (NE) reuptake, while limiting the risks of cardiovasculardisturbances and/or of organ and/or tissue toxicity. More specifically,the mixture of enantiomers in accordance with the invention is intendedto treat depression, chronic fatigue syndrome and urinary incontinence.

Milnacipran (Z(±)-2-(amino methyl)-N,N-diethyl-1-phenyl cyclopropanecarboxamide), a molecule synthesised at the PIERRE FABRE MEDICAMENTResearch Centre (Castres, France), also called TN-912, dalcipran,minalcipran, midalcipran or midalipran is known to be a dual inhibitorof serotonin (5-HT) and norepinephrine (NE) reuptake. Milnacipran andits method of preparation are described in U.S. Pat. No. 4,478,836.Other information relating to milnacipran can be found in the twelfthedition of the Merck Index, as entry n^(o) 6 281.

Dual inhibitors of serotonin (5-HT) and norepinephrine (NE) reuptakecorrespond to a well-known class of antidepressant agents whichselectively inhibit reuptake of both serotonin and norepinephrine. Byway of example, venlafaxine and duloxetine are also dual inhibitors ofserotonin and norepinephrine. Studies have shown that the ratio ofnorepinephrine reuptake inhibition to serotonin reuptake inhibition bymilnacipran is approximately 2:1 (Moret et al., 1985 Neuropharmacology24(12): 1211-1219; Palmier et al., 1989, Eur J Clin Pharmacol 37:235-238).

U.S. Pat. No. 4,478,836 describes the use of milnacipran for thetreatment of disorders of the central nervous system, in particulardepression. Patent application WO01/26623 describes the use ofmilnacipran in association with phenylalanine and tyrosine inindications such as the treatment of fatigue, syndromes associated withpain, chronic fatigue syndrome, fibromyalgia, and irritable bowelsyndrome. Patent application WO01/62236 describes a compositioncontaining milnacipran in association with one or several antimuscarinicagents in a large number of indications including depression.Application WO97/35574 describes a pharmaceutical composition containingmilnacipran and idazoxan as an associated product for usesimultaneously, separately or staggered in time to treat depression andits various forms, as well as disorders in which antidepressants areused. Milnacipran is also indicated for use in the treatment of urinaryincontinence (FR 2 759 290).

Milnacipran exists in the form of two optically active enantiomers: thedextrogyral enantiomer or Z-(1S,2R)-2-(aminomethyl)-N,N-diethyl-1-phenyl cyclopropane carboxamide and the levogyralenantiomer Z-(1R, 2S)-2-(amino methyl)-N,N-diethyl-1-phenyl cyclopropanecarboxamide. In its hydrochloride form, milnacipran (also called F2207)is currently marketed (IXEL, PIERRE FABRE MEDICAMENT, France) in theform of a racemic mixture as a serotoninergic and norepinephrinergicantidepressant agent. F2695 and F2696 designate the dextrogyral andlevogyral enantiomers respectively of milnacipran hydrochloride (F2207):

These two enantiomers can be separated and isolated using proceduresdescribed in the literature (Bonnaud et al., 1985, Journal ofChromatography, Vol. 318: 398-403; Shuto et al., Tetrahedron letters,1996 Vol. 37: 641-644; Grard et al., 2000, Electrophoresis 2000 21:3028-3034; Doyle et Hu, 2001, Advanced Synthesis and Catalysis, Vol.343: 299-302).

The inventors have now performed a pharmacokinetic study in man on theracemate and on the two enantiomers of milnacipran which usesenantiomer-selective assay methods. They have thus demonstrated theabsence of racemisation of the enantiomers in vivo.

Furthermore, although the racemate has been resolved, no analysis of thepharmacological and toxicological properties of the two enantiomers hasbeen performed using modern, currently-available methods such ascardiovascular measurements by telemetry, or genomic analyses forpredictive pharmacotoxicology in vitro.

As with any active substance, antidepressants can induce adverse eventsor certain toxic effects that essentially derive from thepharmacological properties of these drugs, as well as from the dosage,from individual variations in patients (genetic polymorphism,organ-function insufficiency, sex, age) or from drug interactions.Antidepressants are thus the third most common class of productsresponsible for intoxication, after hypnotics and tranquillisers (Noreset al., 1987 Thérapie 42: 555-558). The risk of overdose withantidepressants is serious, since it can lead to death. Among the causesof acute intoxication with antidepressants should be mentionedaccidental ingestion by children (all the more so since certainantidepressants are used in the treatment of enuresis), suicideattempts, accidental overdosage by physicians, concomitant medicationsin elderly patients, age-related physiological and pharmacokineticchanges (cardiac insufficiency, hepatic and/or renal insufficiency . ..) and slowing down of metabolism whether genetic in origin ordrug-induced (enzyme inhibition). After children, the elderly thereforerepresent the second at-risk population among patients treated. Elderlypersons have higher plasma concentrations, related to reduced renaland/or hepatic clearance, and the risks of intoxication are more serious(Meadoer-Woodruffet al., 1988 J. Clim. Psychopharmacol. 8: 28-32).

The adverse side-effects, generally benign, which have been observedduring treatment with milnacipran usually occur within the first or thefirst two weeks of treatment and diminish thereafter, in parallel withimprovement in the depressive episode. The most commonly-reportedadverse events in single-drug therapy or in association with otherpsychotropics are dizziness, hypersudation, anxiety, hot flushes anddysuria. Certain less commonly-reported adverse events are nausea,vomiting, dry mouth, constipation, tremor, palpitations, agitation, andcutaneous eruptions. Moreover, it is known that in patients with ahistory of cardiovascular disease or who concomitantly receive treatmentfor a cardiac condition, milnacipran can increase the incidence ofcardiovascular adverse events (hypertension, hypotension, orthostatichypotension, palpitations). In patients with high blood pressure orhaving heart disease, it is therefore recommended to increase medicalsupervision since milnacipran in the form of a racemic mixture is likelyto increase the heart rate. In those rare cases of overdose observedwith milnacipran (at doses from 800 mg to 1 g) in single-therapy, themain symptoms observed are vomiting, respiratory disturbances andtachycardia (The Vidal Dictionary, 78th Edition, 2002). Another adverseevent occasionally induced by milnacipran is elevated transaminaselevels which may reflect a certain hepatic toxicity.

The at-risk populations that could potentially develop a certain numberof adverse clinical manifestations during or following treatment withmilnacipran are children, the elderly, patients with hepatic and/orrenal insufficiency, patients receiving treatment that induces organand/or tissue toxicity, in particular hepatic or renal toxicity,patients receiving treatment for a heart condition or that inducescardiovascular side-effects, patients with a history of cardiovasculardisease and/or having cardiovascular disorders, especially those withdisorders of cardiac rhythm, of blood pressure (hypo- or hypertensivepatients) and patients suffering from heart disease.

Concerned to prevent, to an ever-greater extent, the occurrence ofpossible side-effects that could constitute a danger, however small, tothe health of patients treated with milnacipran, the inventors have nowdiscovered that, surprisingly and unexpectedly, the dextrogyralenantiomer of milnacipran, which is essentially responsible for theselective inhibitory activity on serotonin and norepinephrine reuptake,induced fewer side-effects of a cardiovascular nature and less organand/or tissue toxicity, especially hepatic, than the racemic mixture. Inparticular, the inventors have discovered that, in dogs, administrationof the dextrogyral enantiomer of milnacipran leads to a lesser increasein heart rate and blood pressure, particularly diastolic blood pressure,than that which can be induced by administration of the racemic mixture.Moreover, the inventors have discovered that the dextrogyral enantiomerof milnacipran (F2695) has a better profile of genomic toxicity than thelevogyral enantiomer of milnacipran (F2696) in an experimental modelusing primary rat hepatocytes. The inventors have also demonstrated thatthe levogyral enantiomer of milnacipran (F2696) has a profile of genomictoxicity similar to that obtained with clomipramine, which is used as areference psychotropic product known for its relative hepatic toxicity.

The object of the present invention is thus the use of a mixture ofenantiomers of milnacipran enriched in the dextrogyral enantiomer,preferentially the substantially-pure F2695 enantiomer, as well as withtheir pharmaceutically-acceptable salts, for the preparation of a drugintended to prevent or to treat disorders or conditions that can bemanaged by double inhibition of serotonin (5-HT) and norepinephrine (NE)reuptake, while limiting the risks of cardiovascular disturbances and/orwhile limiting the risks of organ and/or tissue toxicity.

The term “cardiovascular disturbances” is understood to refer to adversecardiovascular side-effects of the drug administered alone or inassociation with other active substances.

For the purposes of the present invention, the term “side-effect” isunderstood to mean the foreseeable activity of a drug in an area otherthan that for which it is administered, that may be bothersome orundesirable when it limits the use of the drug.

The term “toxicity” is understood to mean the property of a drug toinduce harmful effects on organs or tissue, in particular organs ortissues involved in the metabolism of milnacipran, especially hepaticand/or renal metabolism of milnacipran, and more specifically during thefirst pass of milnacipran in the liver. Preferentially, organ toxicityis cardiac toxicity and the said tissue toxicity is hepatic and/or renaltoxicity.

For the purposes of the present invention, the phrases “while limitingthe risks of cardiovascular disturbances” or “while limiting the risksof toxicity” is understood to mean the fact of preventing these risksfrom increasing significantly in a patient following administration ofthe drug.

For the purposes of the present invention, the term “dextrogyralenantiomer of milnacipran” designates the dextrogyral enantiomer ofmilnacipran, as well as its pharmaceutically-acceptable salts.Preferentially, this is the dextrogyral enantiomer of milnacipranhydrochloride (F2695). “Levogyral enantiomer of milnacipran” designatesthe levogyral enantiomer of milnacipran, as well as itspharmaceutically-acceptable salts (F2696). “Racemic mixture” designatesa 50:50 mixture by weight of the dextrogyral enantiomer of milnacipranand the levogyral enantiomer of milnacipran, as well as theirpharmaceutically-acceptable salts.

For the purposes of the present invention, “mixture of the enantiomersof milnacipran enriched in the dextrogyral enantiomer” signifies amixture of the dextrogyral enantiomer and the levogyral enantiomer ofmilnacipran in which the mass/mass ratio of the dextrogyral enantiomerto the levogyral enantiomer is greater than 1:1. In the mixture of theenantiomers of milnacipran enriched in the dextrogyral enantiomer, themass/mass ratio of the dextrogyral enantiomer to the levogyralenantiomer is advantageously greater or equal to 55:45, moreadvantageous when greater than 60:40, yet more advantageous when greaterthan 65:35, yet more advantageous when greater than 70:30, yet moreadvantageous when greater than 75:25, yet more advantageous when greaterthan 80:20. Produced in a particularly advantageous mode, the mass/massratio of the dextrogyral enantiomer to the levogyral enantiomer isgreater than 82:18, in a more advantageous manner greater than 84:16, inan even more advantageous manner greater than 86:14, in an even moreadvantageous manner greater than 88:12, in an even more advantageousmanner greater than 90:10. Produced in a preferred mode, the mass/massratio of the dextrogyral enantiomer to the levogyral enantiomer isgreater than 91:9, in a more preferred manner greater than 92:8, in aneven more preferred manner greater than 93:7, in an even more preferredmanner greater than 94:6, in an even more preferred manner greater than95:5, in an even more preferred manner greater than 96:4, in an evenmore preferred manner greater than 97:3, in an even more preferredmanner greater than 98:2, in an even more preferred manner greater than99:1, in an even more preferred manner greater than 99.5:0.5. In aparticularly preferred manner, the mixture of enantiomers of milnacipranenriched in the dextrogyral enantiomer is substantially pure, that is tosay, containing approximately 100% dextrogyral enantiomers by weight.

The use of metabolites also enters into the scope of the presentinvention, preferentially the metabolites of milnacipran that are activein vivo, in their Z or E form, and their pharmaceutically-acceptablesalts, such as:

-   -   the hydrochloride of Z-phenyl-1 aminomethyl-2 cyclopropane        carboxylic acid (F1567):

F1567

Molecular mass: 277.7 Characteristics: white crystals Melting point:230° C. Plate chromatography: medium: silica Solvent:Butanol/Ethanol/water (6/2/2) Developer: Ultraviolet and ninhydrineR_(F): 0.6

-   -   phenyl-3 methylene-3-4 pyrrolidone-3 (F1612):

F1612

Molecular mass: 173.2 Characteristics: white crystals Melting point: 70°C. Plate chromatography: medium: silica Solvent: Benzene/dioxane/ethanol(90/25/4) Developer: Ultraviolet and iodine R_(F): 0.46

-   -   the hydrochloride of Z-(para-hydroxyphenyl)-1        diethylaminocarbonyl-1 aminomethyl-2 cyclopropane (F2782):

F2782

Molecular mass: 298.82 Characteristics: white crystals Melting point:250° C. Plate chromatography: medium: silica Solvent:Butanol/Ethanol/water (6/2/2) Developer: Ultraviolet and iodine -ninhydrine R_(F): 0.42

-   -   the oxalate acid of Z-phenyl-1-ethylamino carbonyl-1        aminomethyl-2 cyclopropane (F2800):

F2800

Molecular mass: 308.33 Characteristics: white crystals Melting point:150° C. Plate chromatography: medium: silica Solvent:CHCl₃/methanol/NH₄OH (90/9/1) Developer: Ultraviolet and ninhydrineR_(F): 0.40

-   -   the hydrochloride of Z-phenyl-1 aminocarbonyl-1 aminomethyl-2        cyclopropane (F2941):

F2941

Molecular mass: 226.74 Characteristics: white crystals Melting point:245° C. Plate chromatography: medium: silica Solvent:CHCl₃/methanol/NH₄OH (80/18/2) Developer: Ultraviolet and ninhydrineR_(F): 0.30

These metabolites have, just as milnacipran has, a chiral centre whichconfers optical isomerism on these metabolites that exist in the form ofdextrogyral and levogyral enantiomers. The racemic ratio of the twoenantiomers of the milnacipran metabolite in the mixture of enantiomersis as previously described for the enantiomers of Milnacipran.

The present invention covers therefore these active metabolites, as wellas their pharmaceutically-acceptable salts, in addition to their use asa drug in the treatment of the disorders described in the present patentsuch as depression, pain, fibromyalgia and urinary incontinence. Themetabolites in accordance with the invention are in the form ofracemates or preferentially in the form of a mixture of enantiomersenriched in the most active enantiomer. In a preferable manner, theactive metabolite used comes from the F2695 enantiomer and is thedextrogyral enantiomer of the active metabolite. In a more preferablemanner, this is the dextrogyral enantiomer of the hydrochloride ofZ-(para-hydroxyphenyl)-1 diethylaminocarbonyl-1 aminomethyl-2cyclopropane (F2782). The term “active metabolite” is understood todesignate a derivative of milnacipran metabolised in vitro or in vivoand having the capacity to inhibit reuptake of serotonin and ofnorepinephrine; preferentially, these are F2782, F2941, F2800, F1612 andF1567. For the purposes of the present invention, the active metabolitesin vivo described and claimed for in the present invention include allthe enantiomers, the isomers or the tautomers when the component iscapable of being present in the form of an enantiomer, an isomer or atautomer.

The object of the present invention is therefore the use of a mixture ofenantiomers preferentially enriched in the dextrogyral enantiomer of atleast one metabolite of Milnacipran, preferentially chosen among F2782,F2941, F2800, F1612 and F1567, as well as theirpharmaceutically-acceptable salts, for the preparation of a drugintended to prevent or to treat disorders or conditions that can bemanaged by double inhibition of reuptake of serotonin (5-HT) and ofnorepinephrine (NE), while limiting the risks of cardiovasculardisturbances and/or while limiting organ and/or tissue toxicity, inparticular, cardiac, hepatic and/or renal toxicity.

The use of a mixture of enantiomers of milnacipran enriched in thedextrogyral enantiomer, preferentially the substantially-pure F2595enantiomer, and at least one of its active metabolites, preferentiallychosen among F2782, F2941, F2800, F1612 and F1567, preferentiallyenriched in the dextrogyral enantiomer, for the preparation of a drugintended to prevent or to treat disorders or conditions that can bemanaged by double inhibition of reuptake of serotonin (5-HT) and ofnorepinephrine (NE), while limiting the risks of cardiovasculardisturbances and/or while limiting organ and/or tissue toxicity, inparticular, cardiac, hepatic and/or renal toxicity also enters into thescope of the present invention.

“Pharmaceutically-acceptable salts” designates all salts that retain theefficacy and properties of an active substance and that do not causeside-effects. Such salts may be prepared starting from acids or bases,organic or mineral. Preferentially, these arepharmaceutically-acceptable salts of mineral or organic acids. By way ofexample, but not limited to these, halogen hydrates such as thehydrochloride and the bromohydrate, the fumarate, the maleate, theoxalate, the citrate, the methane sulphonate, the glutamate, thetartrate, the mesylate and their possible hydrates should be mentioned.

For the purposes of the present invention, the term “mixture ofenantiomers” signifies the mixture of enantiomers of milnacipranenriched in the dextrogyral enantiomer, as well as theirpharmaceutically-acceptable salts, and/or the mixture of enantiomers ofat least one of the metabolites of milnacipran, preferentially enrichedin the dextrogyral enantiomer, as well as theirpharmaceutically-acceptable salts.

The mixture of enantiomers in accordance with the invention,preferentially the substantially-pure F2695 enantiomer, is administeredto all types of patients requiring such treatment, whether it be fortherapeutic and/or prophylactic purposes. For therapeutic purposes, theaim is to eradicate or to improve the condition to be treated and/or oneor more related symptoms. For prophylactic purposes, the aim is toprevent the appearance of the condition to be treated and/or of one ormore related symptoms. Nevertheless, the mixture of enantiomers inaccordance with the invention is particularly adapted to populations ofat-risk patients who may be likely to develop certain adverse clinicalmanifestations during or following treatment with milnacipran in theracemic form. These are principally children, the elderly, patients withhepatic and/or renal insufficiency, patients receiving treatment thatinduces hepatic or renal organ and/or tissue toxicity, patientsreceiving treatment for a heart condition, patients receiving treatmentthat induces cardiovascular side-effects, patients with a history ofcardiovascular disease (for example, myocardial infarctus) and/or havingcardiovascular disorders, such as patients with cardiac rhythm disorders(tachycardia, bradycardia, palpitations), patients with blood pressuredisorders (hypo- or hypertensive patients) or patients suffering fromheart disease.

Among the numerous disorders or conditions that have as symptoms cardiacrhythm disorders and for which the present invention is particularlywell-adapted in the treatment of at-risk patients who suffer from them,tachycardia which corresponds to an acceleration of the rhythm of theheart beat (tachycardia is moderate when the heart rate is from 80 to100 beats per minute, severe when it exceeds 100), palpitations,extrasystoles (sporadic, frequent or during myocardial infarctus),auricular fibrillation, flutter and auricular tachysystole, bradycardia,cardiac insufficiency, and myocardial infarctus should be mentioned.

Among the numerous disorders or conditions that have as symptoms bloodpressure disorders and for which the present invention is particularlywell-adapted in the treatment of at-risk patients who suffer from them,arterial hypertension, malignant arterial hypertension, pulmonaryarterial hypertension, portal hypertension, paroxysmal essentialhypertension, hypotension, orthostatic hypotension and intra-cranialhypertension should be mentioned.

Advantageously, those cardiovascular disorders for which the risks canbe limited by the administration of the mixture of enantiomers inaccordance with the invention, and preferentially by the administrationof the substantially-pure F2695 enantiomer, are as follows:

-   -   elevated diastolic and/or systolic blood pressure measured in        millimeters of mercury (mmHg); more specifically, this is an        increase in diastolic blood pressure, and/or,    -   cardiac rhythm disorders, in particular, an increase in the        patient's heart rate.

Systolic blood pressure is the maximal value for blood pressure, and itcorresponds to the moment when the first heart sound is heard in thehumeral artery during measurement of blood pressure. The systole is theinterval of the cardiac cycle during which the heart cavities contract,causing expulsion of the blood. Diastolic blood pressure is the minimalvalue of blood pressure, corresponding to the disappearance of heartsounds in the humeral artery when the cuff of the sphygmomanometer isdeflated during measurement of blood pressure. The diastole is theinterval of the cardiac cycle during which the heart cavities fill withblood. Elevation of systolic and/or diastolic pressure means increasedblood pressure which is characteristic of systemic arterial hypertension(and its variant forms), the symptoms of which may be the following:headache, fatigue, mild sensorial disturbances such as dizziness,buzzing in the ears, palpitations, nosebleed, confusion or drowsiness,cramps, numbness or tingling in the feet and hands. Systemic arterialhypertension (and its variant forms) can lead to serious, indeed fatal,complications: cerebral vascular accidents, left ventricular heartfailure, kidney failure, ischemic heart diseases (myocardial infarctus,angor and their variant forms). According to current guidelines, apatient is considered to have arterial hypertension when his/herdiastolic blood pressure is above 90 mmHg and his/her systolic bloodpressure is above 140 mmHg.

The toxicity for which the risks can be limited by the administration ofthe mixture of enantiomers in accordance with the invention isadvantageously organ toxicity, particularly cardiac toxicity, and/ortissue toxicity, in particular hepatic and/or renal toxicity. Tissuetoxicity may be revealed by the presence of icterus or by laboratorymarkers.

The use of the mixture of enantiomers in accordance with the inventionin veterinary medicine for the treatment of animals, in particularhousehold pets or breeding animals that require such treatment alsoenters into the scope of the present invention.

Because of their pharmacological properties, in particular as dualinhibitors of serotonin (5-HT) and norepinephrine (NE) reuptake, themixture of enantiomers is especially useful in the preparation of drugsintended for preventive and/or curative treatment of a number ofdisorders and conditions (syndromes) described hereinafter, whilelimiting the risks of cardiovascular disturbances and/or while limitingorgan and/or tissue toxicity, in particular cardiac, hepatic and/orrenal toxicity.

Among these disorders or conditions, disorders of the central nervoussystem as defined in <<The Diagnostic and Statistical Manual of MentalDisorders—IV (DSM-IV), 1995 American Psychiatric Association>> should bementioned. By way of example, but not limited to these, the followingdisorders and conditions should be mentioned: depression, in particulardeep depression, resistant depression, depression in the elderly,psychotic depression, depression induced by treatment with interferon,depressive state, manic-depressive syndrome, seasonal depressiveepisodes, depressive episodes related to general health status,depressive episodes related to mood-altering substances, bi-polardisease, schizophrenia, generalised anxiety, morose and marasmic states,stress-related diseases, panic attacks, phobias, in particularagoraphobia, obsessive-compulsive disorders, behavioural disorders,oppositional disorders, post-traumatic stress disorder, depression ofthe immune system, fatigue and accompanying pain syndromes, chronicfatigue syndrome, fibromyalgia, and other functional somatic disorders,autism, disorders characterised by attention deficit due to generalhealth status, attention disorders due to hyperactivity, eatingdisorders, neurotic bulimia, neurotic anorexia, obesity, psychoticdisorders, apathy, migraine, pain and in particular chronic pain,irritable bowel syndrome, cardiovascular diseases and in particularanxiety-depressive syndrome in myocardial infarctus or in hypertension,neuro-degenerative diseases and related anxiety-depressive syndromes(Alzheimer's disease, Huntington's chorea, Parkinson's disease), urinaryincontinence, in particular urinary incontinence related to stress andenuresis, drug addiction and in particular anxiety addiction to tobacco,in particular to nicotine, to alcohol, to narcotics, to drugs, toanalgesics used in weaning-off from these addictive states.

More specifically, the object of the present invention concerns the useof a mixture of enantiomers in accordance with the invention,preferentially the substantially-pure F2695 enantiomer, for thepreparation of a drug intended to treat or to prevent depression ordepressive state while limiting the risks of cardiovascular disturbancesand/or while limiting organ and/or tissue toxicity, in particularhepatic and/or renal toxicity. In the context of the present invention,the term “depression” is understood to refer to a constellation ofsymptoms having, on the one hand, a psychological aspect consisting ofmood disorders with pessimism, moral suffering, thoughts of death orsuicide, mental inhibition, and on the other hand, a physical aspect ofmotor deficit, consisting in particular of a slowdown in motor activity,of appetite disturbances, of constipation, of sleep disturbances and ofweight-control disturbances. Depression therefore corresponds topathological psychological state combining a painful mood-alteration anda reduction in mental and motor activity. The term “depressive state” isunderstood to refer to a mental state characterised by a decline inneuropsychological tonicity, manifesting as lassitude, tendency tofatigue, discouragement and tendency to pessimism sometimes accompaniedby anxiety.

Furthermore, the object of the present invention concerns morespecifically the use of a mixture of enantiomers in accordance with theinvention, preferentially the substantially-pure F2695 enantiomer, forthe preparation of a drug intended to prevent or to treat fibromyalgiaand/or chronic fatigue syndrome while limiting the risks ofcardiovascular disturbances and/or while limiting organ and/or tissuetoxicity, in particular hepatic and/or renal toxicity. Fibromyalgiasyndrome is a chronic syndrome characterised by a feeling of pain andburning with morning stiffness mainly affecting articular andperi-articular fibrous tissues, and by a feeling of deep fatigue.Fibromyalgia includes a constellation of symptoms. The most frequent arenon-restorative sleep, headache, digestive disturbances, depressivestate, muscle spasm, facial pain, numbness etc. Chronic fatigue syndromeis characterised by a state of exhaustion or of fatigue. The most commonsymptoms are a state of weakness, spasms and/or muscle pain, excessiveneed for sleep, fever, angina, memory loss and/or difficultyconcentrating, insomnia, depression.

In addition, the object of the present invention concerns morespecifically the use of a mixture of enantiomers in accordance with theinvention, preferentially the substantially-pure F2695 enantiomer, forthe preparation of a drug intended to prevent or to treat pain and inparticular chronic pain while limiting the risks of cardiovasculardisturbances and/or while limiting organ and/or tissue toxicity, inparticular hepatic and/or renal toxicity. Pain may be associated withvarious disorders and/or wounds. It may be acute or chronic.Epidemiological studies have demonstrated the relations between statesof chronic pain and anxiety depression. Thus, patients suffering fromchronic pain may develop emotional problems that lead to depression,and, in the worst cases, to a suicide attempt. A patient is consideredto be in chronic pain if he/she complains of suffering for a period ofmore than six months. Among the various forms of chronic pain, thefollowed should be mentioned by way of example, but not limited tothese: pain associated with fibromyalgia and/or arising in fibroustissues, muscles, tendons, ligaments and other sites, abdominal pain anddiarrhoea in irritable bowel syndrome, as well as lower back pain.

In addition, the object of the present invention concerns morespecifically the use of a mixture of enantiomers in accordance with theinvention, preferentially the substantially-pure F2695 enantiomer, forthe preparation of a drug intended to prevent or to treat urinaryincontinence and in particular urinary incontinence related to stressand enuresis, while limiting the risks of cardiovascular disturbancesand/or while limiting organ and/or tissue toxicity, in particularhepatic and/or renal toxicity.

Prophylactic and therapeutic treatment of the above-mentioned disordersis achieved by administering to an animal, preferentially to man, atherapeutically-effective quantity of a mixture of enantiomers inaccordance with the invention, preferentially the substantially-pureF2695 enantiomer, alone or in association with at least one other activesubstance. In most cases, this concerns man, however the treatment isalso adapted to animals, in particular breeding animals (livestock,rodents, poultry, fish, . . .) and to domestic animals (dogs, cats,rabbits, horses, . . .).

The mixture of enantiomers, enriched in the dextrogyral enantiomer, ofmilnacipran and/or of at least one of its metabolites, as well as theirpharmaceutically-acceptable salts, as previously described, isadvantageously administered to patients receiving simultaneously,separately or staggered in time at least one other active compound inthe treatment of the above-mentioned disorders.

Preferentially, the object of the present invention also includes, foruse as a drug:

-   -   a) the said mixture of enantiomers enriched in the dextrogyral        enantiomer of milnacipran and/or of at least one of its        metabolites as well as their pharmaceutically-acceptable salts,        and    -   b) at least one active compound chosen among the psychotropics,        in particular antidepressants, and antimuscarinic agents        as associated products for use simultaneously, separately or        staggered in time in the treatment or the prevention of        depression, in particular deep depression, resistant depression,        depression in the elderly, psychotic depression, depression        induced by treatment with interferon, depressive state,        manic-depressive syndrome, seasonal depressive episodes,        depressive episodes related to general health status, depressive        episodes related to mood-altering substances.

The term “psychotropic” is understood to designate a substance ofnatural or artificial origin capable of modifying mental activity andwhose action is essentially exerted on the central nervous system andthe psychological state. Psychotropics are divided into three groups: 1)psycholeptics (hypnotics, neuroleptics and anxiolytics), 2)psychoanaleptics (antidepressants and psychotonics) and 3)psychodysleptics (hallucinogenics).

Preferentially, the said psychotropic is an antidepressant. By way ofexample, but not limited to these, the antidepressant is chosen among(i) monoamine oxidase inhibitors (MAOIs) such as iproniazid, pargyline,selegiline, (ii) 5HT1D-agonists such as sumatriptan, epinephrine andnorepinephrine (alpha and beta sympathomimetics), (iii) tricyclicantidepressants, such as imipramine, clomipramine, (iv) selectiveserotonin reuptake inhibitors (SSRIs) such as fluoxetine, (v) selectivenorepinephrine reuptake inhibitors, such as for example tandamine,fluparoxan, mirtazapine, (vi) serotonin norepinephrine reuptakeinhibitors, such as venlafaxine and duloxetine. By way of example, butnot limited to these, the antimuscarinic agent is chosen amongtolterodine, propiverine, oxybutynin, trospium, darifenacine,temiverine, ipatropium.

Preferably, the object of the present invention also includes for use asa drug:

-   -   a) the said mixture of enantiomers enriched in the dextrogyral        enantiomer of milnacipran and/or of at least one of its        metabolites as well as their pharmaceutically-acceptable salts,        and    -   b) at least one other active substance chosen among the active        compounds inducing organ toxicity and the active compounds        inducing tissue toxicity, in particular hepatic and/renal        toxicity or with one or more active substances intended for        treatment of hepatic or renal insufficiency.        as associated products for use simultaneously, separately or        staggered in time in the treatment or the prevention of        conditions or disorders that can be managed by double inhibition        of serotonin (5-HT) and norepinephrine (NE) reuptake.

Preferably, the object of the present invention also includes, for useas a drug:

-   -   a) the said mixture of enantiomers enriched in the dextrogyral        enantiomer of milnacipran and/or of at least one of its        metabolites as well as their pharmaceutically-acceptable salts,        and    -   b) at least one other active substance chosen among active        compounds inducing cardiovascular side-effects or compounds        given to treat a heart condition.        as associated products for use simultaneously, separately or        staggered in time in the treatment or the prevention of        conditions or disorders that can be managed by double inhibition        of serotonin (5-HT) and norepinephrine (NE) reuptake.

Advantageously, the cardiovascular side-effects induced are thosementioned previously, and more specifically, arterial hypertension,hypotension, cardiac rhythm disorders (tachycardia, bradycardia,palpitations).

The object of the present invention also includes pharmaceuticalcompositions containing the associated products previously described.

In the context of the present invention, the mixture of enantiomers inaccordance with the invention, preferentially the substantially-pureF2695 enantiomer, is advantageously administered, but not in a limitedmanner, via the oral route, the nasal route, the transdermal, rectal,intestinal or parental route, by intramuscular, subcutaneous orintravenous injection, alone or in association with other activesubstances, as previously described.

When administered alone, the mixture of enantiomers in accordance withthe invention, preferentially the substantially-pure F2695 enantiomer,may be administered per se or in the form of a pharmaceuticalcomposition in which the said mixture of enantiomers or of theirpharmaceutically-acceptable salts, is combined or mixed with one orseveral media, pharmaceutically-acceptable excipients and/or diluents,particularly to enhance bioavailability.

When the mixture of enantiomers in accordance with the invention, andpreferentially the substantially-pure dextrogyral F2695 enantiomer ofmilnacipran is administered in association with other active substances,the said mixture and the other active substances may be formulated as amixture or separately in an identical or different form. They may beadministered via the same or a different route.

The pharmaceutical composition in accordance with the invention may beformulated in a conventional manner well-known to the man skill in theart using one or more physiologically-acceptable media includingexcipients, adjuvants and additives such as for example preservatives,stabilisers, wetting agents or emulsifiers. The method of formulationchosen depends on the desired route of administration.

In the context of administration by injection, an aqueous solution isadvantageously used, in particular a physiologically-acceptable buffersolution, such as Hank's solution, Ringer's solution or physiologicalsaline solution. In the context of transdermal administration or via themucous membranes, penetrating agents appropriate to the mucous membraneto be crossed are advantageously used. Such penetrating agents are wellknown to the man skill in the art. In the context of oraladministration, the pharmaceutical compositions in accordance with theinvention are advantageously administered in unit-dose or multiple-doseadministration forms in mixtures containing appropriate pharmaceuticalmedia known to the man skill in the art. Appropriate unit-doseadministration forms include in particular tablets, possibly scored,capsules, powders, granules, oral solutions or suspensions, andaerosols. Appropriate multiple-dose administration forms include inparticular drinkable drops, emulsions and syrups.

In the preparation of tablets, the mixture of enantiomers in accordancewith the invention, preferentially the substantially-pure F2695enantiomer, is formulated with a pharmaceutically-acceptable vehiclesuch as in particular polyvinylpyrrolidone, carbopol gal, polyethyleneglycol, gelatine, talc, starch, lactose, magnesium stearate, gum arabicor their analogues. By way of example, the tablet contains the followingexcipients: calcium hydrogen phosphate dihydrate, calcium carmellose,povidone K30, anhydrous colloidal silicon dioxide, magnesium stearate,talc. The tablets may also be coated, that is to say, covered withseveral coats of various substances such as saccharose in order tofacilitate swallowing or preservation. The coating may also contain dyesor colorants in order to differentiate and to characterise the tabletswith regard to their dosage strength, for example. The tablets may alsobe presented in a more or less complex formulation intended to modifythe rate of release of the active substance. Release of the activesubstance of the said tablet may be rapid, sustained or delayeddepending on the desired absorption. Thus, the mixture of enantiomers inaccordance with the invention, preferentially the substantially-pureF2695 enantiomer, may be prepared in a pharmaceutical form for sustainedrelease obtained according to the process described in patent EP 939626. This pharmaceutical form is presented in the form of multiparticlescontaining a large number of mini-granules and has a certain releaseprofile in vitro.

Release of the mixture of enantiomers in accordance with the inventionmay be delayed and/or controlled by using an implant or by transdermaldelivery, in particular subcutaneous or intramuscular, by intramuscularinjection or by a transdermal patch. The said mixture is thenformulated, in particular, with appropriate hydrophobic or polymericsubstances and ion-exchange resins.

The quantity of the mixture of enantiomers in accordance with theinvention, preferentially the substantially-pure F2695 enantiomer, to beadministered to the patient depends on the condition to be treated, thedesired effect, in particular a therapeutic or prophylactic effect, thehealth status and age of the patient, in particular his/her medicalhistory of cardiovascular disease, the conditions of treatment and themethod of administration of the drug. The quantity required to beadministered for effective therapeutic or prophylactic use in a humanpatient can be determined based on animal models or on data, known tothe man skill in the art, obtained during the treatment of depression inman, for example, using a racemic mixture of Milnacipran.

In the context of therapeutic and/or prophylactic treatment of thedisorders mentioned above, and in particular depression, depressivestates, fibromyalgia, chronic fatigue syndrome, pain, the drug inaccordance with the invention is advantageously administered at dosesfrom 0.01 mg to 10 mg/kg body weight per day in one or more intakes,more advantageously at doses from 0.05 mg to 5 mg/kg body weight per dayin one or more intakes, and even more advantageously at doses from 0.1mg to 1 mg/kg body weight per day in one or more intakes. In aparticularly advantageous manner, administration of the said medicinalproduct at such doses as those defined above is divided into two dailyintakes, preferentially in capsule form. By way of example, the mixtureof enantiomers in accordance with the invention, preferentially thesubstantially-pure F2695 enantiomer, is advantageously administered inthe form of a capsule containing approximately 6.75 mg of activesubstance per capsule, 12.5 mg/capsule, 25 mg/capsule, 50 mg/capsule.

Other characteristics, aims and advantages of the inventions will becomeapparent in the examples that follow. The invention is not limited tothese particular examples which are provided simply by way of exampleand which should be read in comparison with the following figures:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Effects of various treatments on heart rate following singleadministration (mean values)

FIG. 2: Effects of various treatments on mean values of diastolic bloodpressure (mean values over 6 hours following the last intake, after 5consecutive days of treatment).

-   -   ***: p≦0.001 versus deionised water    -   **: p≦0.01 versus deionised water    -   _(p): p≦0.05 versus F2207

FIG. 3: Effects of various treatments on mean values of systolic bloodpressure (mean values over 6 hours following the last intake, after 5consecutive days of treatment).

-   -   ***: p≦0.001 versus deionised water    -   **: p≦0.01 versus deionised water    -   _(p): p<0.05 versus F2207

FIG. 4: Schematic representation of the method of calculation of theToxicity Index. The Toxicity Index is the sum of all up- anddown-regulated genes (in relation to the Induction Factor defined by theuser).

FIGS. 5A, 5B, 5C: MTT assay on primary rat hepatocytes.

The concentrations are expressed in μM

EXAMPLES Example N^(o) 1 Pharmacokinetic Studies on Milnacipran and onits Enantiomers

Pharmacokinetic studies on milnacipran (F2207) and on its enantiomers(F2695 and F2696) were performed in various animal species and in man.

In animals, the pharmacokinetics of each enantiomer were studiedfollowing administration of the racemate or of one single enantiomer.Plasma concentrations of the F2695 and F2696 enantiomers areapproximately equivalent in the animal species tested (monkey and rat).

A pharmacokinetic study in man involving 12 healthy subjects wasperformed by administering the racemate or one of the two enantiomersalone. It was shown that the pharmacokinetic profile of each enantiomeris independent of whether it was administered separately or in the formof the racemate, indicating the absence of interaction between theenantiomers (Table 1).

TABLE 1 Table of the main pharmacokinetic variables of milnacipran(F2207) and its two enantiomers, F2695 and F2696. F2207 Dose (50 mg)F2695 (D) F2696 (L) administered (mg) F2695 (D) F2696 (L) (25 mg) (25mg) Cmax (nmol · l⁻¹) 214 179 216 212 Tmax (hours) 3.42 2.87 3.08 2.21AUC 0->^(∞) 2896 1563 2869 1543 (nmol · h · l⁻¹) T½(hours) 9.28 5.759.38 5.58 Cmax: Maximal plasma concentration directly estimated based onexperimental data Tmax: Time to reach maximal plasma concentration AUC0->^(∞): Area under the curve for plasma concentrations in relation totime extrapolated to infinity T½: Terminal half-life of decrease inplasma concentrations

These findings indicate that no biotransformation of the F2695 or F2696enantiomers was detected in the species studied.

Example N^(o) 2 Biochemical Studies of Milnacipran and of itsEnantiomers

The two enantiomers (F2695 and F2696) of milnacipran (F2207) werestudied in vitro on uptake of norepinephrine and serotonin as well as onbinding of paroxetine in the rat brain.

The presence of an asymmetrical carbon in the chemical structure ofmilnacipran lead to performance of a chiral study on the molecule. Inorder to study the various isomeric forms, the two enantiomers, F2695(Zd) and F2696 (Z1), were separated starting from F2207 in its racemicconfiguration (Z d1), and subjected to tests on uptake of monoamines,norepinephrine and serotonin, and on paroxetine binding.

2.1. Materials and Methods

2.1.1. Norepinephrine Uptake by a Homogenate (P₂) of Rat Hypothalamus.

Preparation of P2

Male Sprague-Dawley rats, from 200 to 300 g, were stunned anddecapitated, and the hypothalami were rapidly removed. Two hypothalamiare homogenised in 4 ml of sucrose 0.32 M on a Potter S by 16 completepasses back and forth at 800 rpm, then centrifuged for 10 min at 1,000 gto eliminate cell debris. The supernate is centrifuged for 20 min at10,000 g and the P₂ thus obtained is recovered in 4 ml of sucrose 0.32M, and homogenised on a Dounce.

Uptake

³H-(1)-NE: 13 Ci/mmol (Amersham) is used.

Uptake takes place in a phosphate buffer (containing 8 g of NaCl, 1.21 gof K₂HPO₄ and 0.34 g of KH₂PO₄ per liter) pre-oxygenated 30 min beforeuse with a mixture of O₂/CO₂ (95%/5%).

In 5-ml plastic tubes placed in a water bath at 37° C., the followingare introduced:

-   -   100 μl of buffer or inhibitor,    -   700 μl of buffer (containing 25 μM pargyline),    -   100 μl of P₂.

After temperature balance, the reaction begins by the addition of 100 μlof ³H-NE, 50 nM final concentration.

Exactly 10 min later, the reaction is stopped by adding 2.5 ml ofchilled buffer and filtering through GF/F filters. The tube is thenrinsed once and the filter once with 2.5 ml of chilled buffer. Thefilter is then introduced into a Beckman mini-vial and, after adding 3ml of Instagel (Packard) liquid scintillator, radioactivity is measuredwith a Tricarb Packard scintillation counter.

Non-specific uptake (NS) is measured as the presence of DMI 10⁻⁵ M.

The percentage of inhibition is calculated using the formula:

$\frac{\left( {{{total}\mspace{14mu}{uptake}} - {NS}} \right) - \left( {{{uptake}\mspace{14mu}{in}\mspace{14mu}{the}\mspace{14mu}{presence}\mspace{14mu}{of}\mspace{14mu}{inhibitor}} - {NS}} \right)}{\left( {{{total}\mspace{14mu}{uptake}} - {NS}} \right)}$

The IC₅₀ is determined graphically on the mean curve of percentage ofinhibition (4 assays) in relation to the log of the concentration ofinhibitor.

2.1.2. Serotonin Uptake

The method was developed following that of Gray and whittaker (1962, J.Anat., 96: 79-97). After homogenisation of brain tissue in a sucrosesolution, the presynaptic terminals break away from the axon and closeto form synaptosomes obtained by subcellular fractionation.

Male Sprague-Dawley (Janvier) rats weighing 180-200 g were used. Aftersacrifice of the animal, the hypothalamus was removed, weighed andhomogenised on a Dounce in 0.32 M sucrose at 0° C.

This homogenate was centrifuged for 10 min at 1,000 g (2,400rpm-Hettich, Rotenta). The supernate was recovered and centrifuged for20 min at 10,000 g (8,000 rpm-Beckam, model n^(o) J2-21 M: J14 rotor).The residue (called the P₂ fraction) was recovered in sucrose at aconcentration of 50 mg/ml.

The following were incubated for 5 min at 37° C.:

-   -   350 μl of chilled buffer (NaCl 136 mM, KH₂PO₄ 2.4 mM, K₂HPO₄ 6.9        mM, pH 7.2) pre-oxygenated 30 min before,    -   50 μl of membranes (5 mg/ml finally),    -   50 μl of citalopram (10⁻⁵ M finally) for non-specific uptake,    -   50 μof ³H-5-HT (50 nM finally) (NEN, France, 28.4 Ci/mmol).

Exactly 5 min after the start of incubation, the reaction was stopped byvacuum filtration on Whatman GF/F filters (predilution with 2.5 ml ofchilled buffer then rinsing with 3 times 2.5 ml).

The radioactivity collected on the filter was measured (Packard Tricarb4640) by liquid scintillation with Emulsifier-Safe (Packard).

The IC₅₀ were determined by transposing the percentages of inhibitiononto a graph in relation to the log of the product concentration (6concentrations in duplicate).

2.1.3. Paroxetine Binding

Male Sprague-Dawley rats (Janvier) weighing 180-200 g were used. Thehypothalami of several rats were collected and homogenised in 5 ml ofchilled buffer (50 mM Tris-HCL, 120 mM NaCl, 5 mM KCl, pH 7.5) on aDounce, and the homogenate was centrifuged at 30 000 g (27 000rpm-Beckman. L5-50E, T40 rotor) for 10 min. The residue obtained wasrecovered in 5 ml of buffer and re-centrifuged under the sameconditions. The new residue was recovered in the same buffer and finallyre-homogenised on a Dounce at a tissue concentration of 10 mg/ml. Themembrane suspension (100 μl) was incubated with 3H-paroxetine (NEN,France, 28.6 Ci/mmol) at a concentration (final) of 0.1 nM, at 20° C.,in a final volume of 1 ml for 2 hr. After 2 hr incubation, the reactionwas stopped by vacuum filtration on Whatman GF/F filters pre-treated ina 0.05% solution of polyethylenimine 30 min beforehand (prediluted with4 ml of chilled buffer, then the tube was rinsed with 2 times 4 ml).Radioactivity was measured by liquid scintillation spectrometry(Packard, Tricarb 4640) using Emulsifier-Safe (Packard) as thescintillating agent.

Specific ³H-paroxetine binding was defined as the difference betweentotal binding and that remaining in the presence of 10 μM of fluoxetine.

The IC₅₀ were determined by transposing the percentages of inhibitiononto a graph in relation to the log of the concentration of the product(6 concentrations in duplicate).

2.1.4. Products Used

-   -   F2207: batch no 10-CTN3 Key P118    -   F2695: batch no PL-1-205    -   F2696: batch no PL-1-204C.        2.2. Results

The effects of F2207 and of its two enantiomers on uptake ofnorepinephrine and serotonin and on paroxetine binding are shown on agraph with the percentage of inhibition in relation (%) on the ordinateand the concentration (M) of F2207, F2695 and F2696 on the apsis (datanot shown). The values for the percentages of inhibition correspondingto each product, tested in duplicate, are mean results of four separateexperiments.

The values of the IC₅₀ for the three products were determined on thebasis of these curves and are shown in table 2.

TABLE 2 Inhibition of ³H-norepinephrine and ³H-serotonin uptake and³H-paroxetine binding. IC50 (M) Uptake ³H-Paroxetine Compounds³H-Norepinephrine ³H-Serotonin Binding F2695 1.5 × 10⁻⁸ 4.6 × 10⁻⁸  6.0× 10⁻⁸  F2207 3.0 × 10⁻⁸ 15 × 10⁻⁸ 13 × 10⁻⁸ F2696  75 × 10⁻⁸ 60 × 10⁻⁸70 × 10⁻⁸

The three compounds were active in these three pharmacological assays,however differences were present:

in norepinephrine uptake:

F2695 was two times more active than F2207.

F2695 was 25 time more active than F2696.

in serotonin uptake:

F2695 was 3 times more active than F2207.

F2695 was 12 times more active than F2696.

in paroxetine binding:

F2695 was 2 times more active than F2207.

F2695 was 10 times more active than F2696.

The three compounds were active in these pharmacological assays withhowever a lesser activity for the levogyral form (F2696) and theracemate (F2207). The dextrogyral form of milnacipran (F2695) was 2 to 3times more active than F2207.

Example N^(o) 3 Comparative Activity of Milnacipran (F2207) and of itsActive Enantiomer (F2695) by the Oral Route on Heart Rate and BloodPressure in the Waking Dog

3.1. Introduction

This study was designed to study the effects of F2207 (batch n^(o)PHA343) and of F2695 (batch n^(o) PL-I-221) a) on heart rate after asingle administration by the oral route, and b) on systolic anddiastolic blood pressure after repeated administration for 5 days by theoral route in dogs.

This study was conducted at equally pharmaceutically-active doses ofF2695 in 6 dogs equipped with implants (Data Sciences International)allowing for data on heart rate and blood pressure parameters to becaptured by telemetry. The animals were allocated to 3 treatment groups:

-   -   group 1 (control) treated with deionised water,    -   group 2 treated with F2207 at a dose of 20 mg/kg/day,    -   group 3 treated with F2695 at a dose of 10 mg/kg/day.        3.2. Methodology        Given the small number of equipped animals available, and in        order to form 3 treatment groups comprising 6 animals each, the        study was carried out in three series separated by a wash-out        period with re-initialisation of the probes. Each series        comprised 2 phases as follows:    -   the first phase of 5 days during which all the animals were        treated with deionised water in order to adapt them to        containment and to oral treatment by gavage with stomach tubing,    -   the second phase of 5 days during which the animals received        their respective treatment.

The treatment regimen and associated schedule were as follows:

-   -   16 Sep. 2002 (D-7): reception of animals in the study,    -   18 to 22 Sep. 2002 (D-5 to D-1): adaptation treatment with        deionised water for all the animals,    -   23 to 27 Sep. 2002 (D1 to D5): 1^(st) treatment series: the        animals received their respective treatment,    -   28 Sep. to 1 Oct. 2002 (D6 to D9): wash-out period,    -   2 to 6 Oct. 2002 (D10 to D14): adaptation treatment with        deionised water for all the animals,    -   7 to 11 Oct. 2002 (D15 to D19): 2^(nd) treatment series: the        animals received their respective treatment,    -   12 to 15 Oct. 2002 (D20 to D23): wash-out period,    -   16 to 20 Oct. 2002 (D24 to D28): adaptation treatment with        deionised water for all the animals,    -   21 to 25 Oct. 2002 (D29 to D33): 3^(rd) treatment series: the        animals received their respective treatment.

The allocation of animals to the various groups and the associatedtreatment are shown in table 3. The overall experimental plan isdescribed in table 4.

TABLE 3 Table of allocation of dogs to the various groups and associatedtreatment. Definitive numbering of animals and associated treatmentProvisional 1^(st) treatment 2^(nd) treatment 3^(rd) treatment numberingseries series series of animals (D1 to D5) (D15 to D19) (D29 to D33) dogn° 102 dog n° 3 dog n° 9 dog n° 13 F2207 (20 mg/kg/d) F2207 (20 mg/kg/d)deionised water dog n° 103 dog n° 1 dog n° 11 dog n° 17 deionised waterF2695 (10 mg/kg/d) F2695 (10 mg/kg/d) dog n° 104 dog n° 4 dog n° 8 dogn° 18 F2207 (20 mg/kg/d) deionised water F2695 (10 mg/kg/d) dog n° 106dog n° 2 dog n° 12 dog n° 16 deionised water F2695 (10 mg/kg/d) F2207(20 mg/kg/d) dog n° 109 dog n° 5 dog n° 7 dog n° 15 F2695 (10 mg/kg/d)deionised water F2207 (20 mg/kg/d) dog n° 110 dog n° 6 dog n° 10 dog n°14 F2695 (10 mg/kg/d) F2207 (20 mg/kg/d) deionised water Nota: accordingto the initial randomisation scheme, each animal was to receive adifferent treatment in each series. An error committed on D15 forced usto revise the randomisation. The animal bearing provisional ID n° 102was in fact presented by mistake and treated with F2207, this animaltherefore received treatment with F2207 twice. In order to maintain thesame number of animals in each treatment group, the animal bearingprovisional ID n° 103 also received the same treatment twice, F2695.

TABLE 4 Overall experimental plan for the telemetry study on the effectsof milnacipran and of F2695 administered orally for 5 days in consciousdogs. GROUP NUMBER 1 2 3 ANIMALS Number 6 6 6 Identification1-2-7-8-13-14 3-4-9-10-15-16 5-6-11-12-17-18 TREATMENT IdentificationDeionised water F2207 F2695 Dose — 20 mg/kg/day 10 mg/kg/day Route oralVolume 5 ml/kg Frequency/Duration daily administration/5 days

The effects of the various treatments on heart rate were analysed aftersingle administration. The analysis concerns the following data-capturetimes:

-   -   prior to single administration,    -   every 30 minutes over 6 hours following single administration.

The effects of the various treatments on blood pressure were analysed atthe steady state, on D5, D29 and D33(final effective day of treatmentfor each series). The analysis concerns the following data-capturetimes:

-   -   prior to treatment,    -   every 30 minutes over 6 hours following treatment.        3.3. Results        3.3.1. With regard to heart rate, a Tukey test was performed for        each of the 13 experimental times.

The following observations were made in comparison with the controlanimals receiving deionised water (FIG. 1):

-   -   § a significant increase in heart rate for half of the        experimental times (6/12) after single administration of F2207        (20 mg/kg/day), particularly marked one to two hours after        treatment (p≦0.01 at 1.5 and 2.0 hours after treatment),        followed by a slow decline, still present and significant        however 5.5 hours after treatment (p≦0.01).    -   § the absence of any significant increase in heart rate for        almost all the experimental times (11/12), after single        administration of F2695 (10 mg/kg/day); one weak statistical        significance was observed at 1.5 hours (p≦0.05).

3.3.2. With regard to blood pressure, one mean value for diastolic bloodpressure (FIG. 2 and table 5), as well as one mean value for systolicblood pressure (FIG. 3 and table 6) were calculated for each dog and forthe 6 hours following the final treatment, after 5 consecutive days ofadministration. These mean blood pressure values were analysed by ANOVAfollowed by a Tukey test when ANOVA permitted such a test (data notshown).

The following were observed:

-   -   a significant increase (p<0.001) in diastolic blood pressure        after repeated administration of F2207 for 5 days (20 mg/kg/day)        or of F2695 (10 mg/kg/day) compared to treatment with deionised        water,    -   a significant difference (p<0.05) in the mean diastolic blood        pressure value after repeated administration of F2207 (20        mg/kg/day) for 5 days compared to the mean diastolic blood        pressure value after repeated administration of F2695 (10        mg/kg/day)    -   no significant effect on systolic blood pressure; it should be        noted however that the values for sBP after repeated        administration of F2695 for 5 days are close to the sBP        following treatment with deionised water.

Individual diastolic and systolic blood pressure data are shown intables 5 and 6 respectively.

TABLE 5 Individual diastolic blood pressure data DIASTOLIC BLOODPRESSURE (dBP expressed in mmHg) Individual data after repeatedadministration for 5 consecutive days GROUP 1 2 3 TREATMENT VEHICLEF2207 (20 mg/kg/d) F2695 (10 mg/kg/d) Animal N° 1 2 7 8 13 14 M SEM 3 49 10 15 16 M SEM 5 6 11 12 17 18 M SEM Time before 79 77 73 77 101 76 814 112 89 93 88 86 91 93 4 73 89 80 71 76 78 78 3 treatment Time aftertreatment(h) 0.50 84 76 70 63 80 70 74 3 103 106 96 92 88 87 95 3 91 9199 90 108 85 94 3 1.00 82 84 77 72 72 76 77 2 130 117 113 113 90 106 1125 112 96 75 97 87 96 94 5 1.50 102 81 79 75 82 68 81 5 131 127 137 96100 91 114 8 109 83 88 97 87 112 96 5 2.00 83 75 71 98 77 75 80 4 123113 99 88 107 109 107 5 115 88 93 95 84 109 97 5 2.50 85 75 75 84 85 7981 2 137 111 116 101 115 107 115 5 111 88 97 89 92 107 97 4 3.00 91 9599 85 79 84 89 3 121 118 112 116 106 92 111 4 104 91 96 96 100 106 99 23.50 83 72 78 73 77 65 75 3 120 106 133 116 103 103 114 5 96 106 94 10777 103 97 5 4.00 81 79 75 77 82 68 77 2 133 114 105 111 110 103 113 4125 91 99 108 80 109 102 6 4.50 82 76 91 84 113 85 89 5 135 110 126 109104 108 115 5 103 104 92 100 85 108 99 3 5.00 97 79 67 95 81 82 84 5 116120 98 97 97 105 106 4 126 100 92 95 110 102 104 5 5.50 94 80 70 ND 8582 82 4 103 107 115 106 92 93 103 4 88 86 105 98 89 99 94 3 6.00 83 7482 82 78 77 79 1 115 133 120 104 103 104 113 5 101 113 98 105 109 108106 2 Mean dBP after 87 79 78 81 83 76 81 2 122 115 114 104 101 101 1104 107 95 94 98 92 104 98 2 treatment ND: not determined

TABLE 6 Individual systolic blood pressure data SYSTOLIC BLOOD PRESSURE(sBP expressed en mmHg) Individual data after repeated administrationfor 5 consecutive days GROUP 1 2 3 TREATMENT VEHICLE F2207 (20 mg/kg/d)F2695 (10 mg/kg/d) Animal N° 1 2 7 8 13 14 M SEM 3 4 9 10 15 16 M SEM 56 11 12 17 18 M SEM Time before 139 141 120 157 172 138 145 7 188 164176 149 130 169 163 8 136 141 138 130 134 149 138 3 treatment Time aftertreatment(h) 0.50 135 132 119 131 149 138 134 4 158 154 152 128 126 129141 6 135 129 140 135 160 139 140 4 1.00 134 158 129 144 141 143 142 4180 167 157 150 126 130 152 9 159 135 124 148 131 143 140 5 1.50 158 151145 150 153 137 149 3 186 181 189 129 136 138 160 12 164 119 138 158 127156 144 8 2.00 138 136 145 173 151 144 148 5 171 160 146 122 140 163 1507 168 125 135 141 127 156 142 7 2.50 142 143 145 159 160 148 150 3 195168 168 144 153 161 165 7 165 124 142 141 134 154 143 6 3.00 149 167 162163 150 154 158 3 173 177 164 157 141 146 160 6 156 131 145 144 151 157147 4 3.50 135 129 149 154 153 137 143 4 165 153 184 167 139 155 161 6146 147 141 169 123 156 147 6 4.00 142 143 149 166 164 144 151 4 180 157151 154 150 153 158 5 180 132 145 160 124 164 151 9 4.50 137 140 159 170190 152 158 8 184 161 180 155 145 168 166 6 158 151 138 163 131 163 1515 5.00 150 146 127 177 160 145 151 7 161 171 146 141 139 166 154 6 182144 137 150 162 158 156 6 5.50 153 149 132 ND 148 144 145 4 151 154 173152 132 155 153 5 142 127 152 153 141 153 145 4 6.00 146 144 151 176 146143 151 5 158 192 171 154 148 172 166 7 156 170 148 159 160 166 160 3Means BP 143 145 143 160 155 144 148 3 172 166 165 146 140 153 157 5 159136 140 152 139 155 147 4 after treatment ND: not determined3.4. Conclusion

Under the experimental conditions of the present study by oraladministration in the waking dog equipped with a telemetric device:

-   -   on single administration and compared to the control group, the        increase in heart rate was weakly significant and fleeting with        F2695 at a dose of 10 mg/kg/day, clearly significant and lasting        with F2207 at the equally pharmaceutically-active dose of 20        mg/kg/day,    -   F2695, at a dose of 10 mg/kg/day, did not induce any        statistically significant change in mean systolic blood pressure        over the 6 hours following the final treatment, at the steady        state after repeated administration for 5 days,    -   a statistically significant difference was evidenced in mean        diastolic blood pressure over the 6 hours following the final        treatment, at the steady state after repeated administration for        5 days, between the active F2695 enantiomer (98±2 mm Hg) and the        F2207 racemic at equally pharmaceutically-active doses (110±4 mm        Hg).

These differences clearly demonstrated greater cardiovasculartolerability of the active F2695 enantiomer.

Example 4 Genomic Test of Predictive Toxicology In Vitro

4.1. Materials and Methods

The F2695 and F2696 compounds, enantiomers of the racemic moleculeF2207, as well as clomipramine, a reference product, (coded C218 in thestudy) were assessed in the present study. The two enantiomers, F2695and F2696, were first assessed in a preliminary cytotoxicity text(MTT-assay) on primary rat hepatocytes, in order to select the threeconcentrations to be used in the final test.

After treatment of the primary rat hepatocytes in culture, the RNA wasextracted in order to generate labelled complementary-DNA probes whichwere then hybridised on a membrane containing 682 alternatively-splicedfragments specific to cell stress. A Toxicity Index was obtained foreach of the products by comparing the hybridisation profile of thetreated cells with that of the untreated cells.

4.1.1. Purpose and Aim of the Study

Safe-Hit is a genomic test for predictive toxicopharmacology that issensitive, robust, reliable, rapid and sure, enabling products to becompared and ranked on the basis of optimised assessment of their toxicpotential.

Safe-Hit uses technology, the property of EXONHIT (DATAS™: DifferentialAnalysis of Transcripts with Alternative Slicing), that permitsisolation and, consequently, cloning of splicing events that result froma given biological state, in comparison with a control condition. Thisallows mRNA isoforms, differentially expressed depending the biologicalconditions, to be isolated.

Safe-Hit allows molecules within a chemical series to be rankedaccording to a Toxic Index, determined after the following basic steps(systematically performed in duplicate for each product):

-   -   treatment of the cells lines with the various products at three        different concentrations, deduced from a preliminary        cytotoxicology test (MTT-assay): a reference concentration        corresponding to 80% cell viability, a concentration 10-fold        higher—when possible—and a concentration 10-fold lower,    -   preparation of total RNA and of the corresponding radio-labelled        cDNA probes,    -   hybridisation of the cDNA probes: the Safe-Hit macroarray        contains 682 independent clones, corresponding to alterations in        gene splicing induced by overexpression of WTp53 (p53 is the        most ubiquitous “mediator” of cell stress, chosen for the        development of this methodology),    -   acquisition and determination of the Toxicity Index.        4.1.2. Cells

The cells used in the study (preliminary MTT-assay of cytotoxicity andthe main test) are cryopreserved hepatocytes from Sprague-Dawley rats inprimary culture (batches Hep184005 and Hep184006-Biopredic), culturedunder standard conditions.

4.1.2.1 Culture Medium

-   -   thawing medium: Leibovitz 15 medium with glutamax 1, to which        were added 100 IU/ml of penicillin, 100 μg/ml of streptomycin        and 0.6 M of glucose (batch MIL 210009-Biopredic),    -   seeding medium: Williams E medium with glutamax 1, to which were        added 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 4        μg/ml of bovine insulin and 10% v/v foetal calf serum (batch MIL        260005)-Biopredic),    -   incubation medium: Williams E medium with glutamax 1, to which        were added 100 IU/ml of penicillin, 100 μg/ml of streptomycin, 4        μg/ml of bovine insulin and 50 μM of hydrocortisone        hemisuccinate (batch MIL 260009-260007-Biopredic).        4.1.2.2 Culture Conditions

37° C., CO2 atmosphere (5%), relative humidity (95%).

4.1.2.3 Culture Procedure

Cell toxicity test Main study Cells were seeded on the day of treatmentSeeding density 35 000 cells/well (96 1.5 million cells per 30 mm wellsper plate) plate Medium volume 0.1 ml 3 ml4.1.3. Cytotoxicity Test

The cytotoxicity test (MTT-assay) detects live cells by use of acalorimetric reaction that reveals the integrity of cell respirationimplying activity of the mitochondria. MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), solublein water, is transformed by splitting, under the effect of amitochondiral enzyme in live cells, into insoluble purple formazan.Formazan is solubilised in an organic solvent and the solution obtainedcan be measured by spectrophotometry. The absorbance measured isproportional to the number of surviving cells.

The cells are put into contact with the product to be tested at 5different concentrations (0-1-10-25-50 and 100 μM) for 16 hours.

After this period of exposure, an MTT solution (0.5 mg/ml in theincubation medium of the primary hepatocytes) is added for 3 hours.After solubilisation of the formazan crystals, the multi-well plates areread with a spectrophotometer at 500 nm in order to determine thepercentage of cell viability.

4.1.4 Main Genomic Pharmacotoxicology Test

The main study is performed in duplicate, using seeded cultures exposedto each product in order to enhance consistency between the experimentsand to validate the results obtained.

4.1.4.1 Cell Seeding and Treatment

The cells are seeded and cultured for 16 hours with each product, at thethree concentrations chosen on the basis of the preliminary MTT-assay;two controls (untreated cells in solvent alone) are added to the series.

4.1.4.2 Total RNA Extraction and Assay

After treatment, the RNA is extracted and analysed as follows:

-   -   collection of cells and centrifugation,    -   extraction performed with a ready-to-use phenol reagent        (Trizol-batches 1106266 and 1121067-Invitrogen) according to the        manufacturer's protocol,    -   solubilisation of the RNA in water,    -   RNA assay by spectrophotometry (optical density measured at 260,        280 and 300 mm),    -   verification of the quality of the RNA using Agilent.        4.1.4.3 Preparation of the cDNA Probes

The cDNA probes are prepared by reverse radio-active transcription(alpha dATP ³³P-Amersham). The radio-active cDNA is quantified (InstantImager-Packard) to ensure that the probes are active.

4.1.4.4 Hybridisation on the Safe-Hit Membrane

The 682 DATAS clones (alternately-spliced patterns) are placed on theSafe-Hit membranes, made of pre-cut nylon (Q-BIOgene), with the aid of aQ-Pix apparatus (GENETIX). The DNA probes are hybridised on themembranes overnight and the membranes are washed.

4.1.1.5. Preparation of the cDNA Probes:

-   -   matrix: 5 μg of total RNA (for each treatment series and for        each concentration),    -   primer: 100 ng of oligo-dTV oligonucleotide, for the 1st and 2nd        hybridisations in rats (batch 12.00, Invitrogen),    -   main mixture:        -   10 μl of First Strand 5× Premier buffer (batch            1131226-Invitrogen)        -   1 μl of dCTP+dGTP+dTTP 20 mM (batch 1105201-Invitrogen)        -   1 μM of ATP 120 μM (batch 1105201-Invitrogen)        -   5 μl of Dithiotreitol (DTT) 0.1 M (batch 133609-Invitrogen)        -   1 μl of Out 40 U RNase (batch 1113345-Invitrogen)        -   5 μl of ³³P dATP 3 000 Ci/mmol 10 mCi/μl (batch            B0239-Amersham)        -   4 μl of Superscript II (batch 1137806-Invitrogen)        -   1 μl of glycogen (batch 1129328-Invitrogen)

Procedure:

Incubate the RNA and the oligo-dTV at 70° C. for 10 minutes then placeit on ice. Add 27 μl of MasterMix and incubate at 43° C. for 1 h than at50° C. for 15 minutes. Add 20 μl of water, then 20 μl of EDTA 50 mM,then 4 μl of NaOH 10N. Incubate for 20 minutes at 65° C. then place onice. Quantification: Instant Imager, Packard: 1 μl of reaction mixture,add 8 μl of acetic acid, 100 μl of isopropanol and 1 μl of glycogen (20μg/μl). Incubate at −20° C. for 20 minutes, centrifuge for 20 minutes at13000 rpm at 4° C. Reconstitute as a suspension with 200 μl of water,quantification: Instant Imager, Packard: 1 μl of reaction mixture.

Media and buffers Common solutions: Washing buffer 1: 20X SSC(Invitrogen) 2X SSC 50X Denhardt's 50% (w/v) Dextran Sulphate (ICN) 20%SDS (v/v)(Quantum biotech.) 10 mg/ml DNA from salmon sperm (Q-Biogene)Prehybridisation buffer: Washing buffer 2: 6X SSC 2X SSC 10X Denhardt's0.1% SDS 10% Dextran Sulphate 0.5% SDS H₂O Hybridisation buffer: Washingbuffer 3: 5X SSC 0.5X SSC 5X Denhardt's 0.1% SDS 0.1% SDS H₂O Washingbuffer 4: 1X SSC 0.1% SDS

Prehybridisation:

-   -   Aliquot 5 ml of prehybridisation buffer in the hybridisation        tubes, add the corresponding volume of salmon-sperm DNA for a        final concentration of 100 μg/ml, soak the membranes in 5×SSC,        place the membrane in the hybridisation tube and prehybridise        for 2 hours at 65° C.

Hybridisation:

-   -   Remove the prehybridisation buffer and rinse with 10-20 ml of        5×SSC, remove the 5×SSC, replace with 5 ml of        buffer+salmon-sperm DNA, denature the RT probes for 5 min at 95°        C., then place on ice for 1 minute, centrifuge to reconstitute,        then recover the appropriate volume of denatured RT probes in        the tube (100,000 to 200,000 cpm/ml), incubate overnight at 55°        C.

Washing:

-   -   Rinse the membranes with 10-20 ml of washing buffer 1, remove        the buffer and replace it with 50 ml of washing buffer 2,        incubate for 30 min at 55° C., then remove and replace while        washing with buffer 4, incubate for 30 mn at 55° C., then pour        off the final washing buffer, remove the membranes from the        tubes, place them on a cassette and allow acquisition to        continue for 3 hours.        4.1.4.5 Acquisition and Analysis of the Image

The membranes are placed on a screen (FX Imaging ScreenK-Bio-rad) for 3hours. The film is then read using a Personal Molecular Imager FX(Bio-rad). The image is analysed using the Safe-Hit Reader Software(COSE).

4.1.4.6 Calculation of the Toxicity Index

All the data are transferred to an automatic calculation programme thatnormalises the various membranes and calculates a Toxicity Index, equalto the sum of the number of up- and down-regulated genes for a givencompound at a given concentration, in comparison with the results of theuntreated controls. The results of the two Safe-Hit analyses are thencompared and combined to assess the potential toxicity of the variouscompounds tested. Two parameters that can be modified by the user areinvolved in the calculation of the Toxicity Index:

-   -   Background Threshold (BT) smoothes out weak signals, close to        background noise and not attributable to significant gene        expression. This therefore determines the threshold of        detection;    -   Induction Factor (IF) is determined as the multiplication        factor, versus the control samples, for the clones to be up- or        down-regulated. The value of this parameter is usually 2 or less        than 2 in order to obtain relevant results. Progressively        increasing the IF value selects those clones that are more and        more strongly up- or down-regulated.

The procedure for calculating the Toxicity Index was developed bycomparing the reference profiles (R: untreated cells) with anexperimental profile (E) and goes through the following steps (see FIG.4 for a schematic overview of the procedure):

-   -   transformation of all the values obtained into log values,    -   calculation of the mean log value for each of the duplicate        assays (M_(iR) et M_(iE)),    -   creation of a matrix with M_(iR)-M_(iE) for all the signals        (=D_(i)),    -   normalisation of the individual M_(iE) values by subtracting        from M_(iE) the median of the 14 proximal values of Di        (=NM_(iE)),    -   comparison of the normalised values with the reference values        (C_(i)=NM_(iE)−M_(IR)),    -   exponential transformation of C_(i)(=F_(i)),    -   comparison of F_(i) with the Induction Factor chosen by the        user:        -   if F_(i)>IF, the gene is considered to be up-regulated,        -   if 1/IF<F_(i)<IF, the gene is considered to be without            change,        -   if F_(i)<1/IF, the gene is considered to be down-regulated.            4.2. Results of the MTT-Assay

These assays were performed in triplicate on primary rat hepatocytesexposed for 16 hours.

Clomipramine, referred to as C218, showed marked toxicity at 100 μMsince no cell viability was observed after exposure of the cells for 16hours. Conversely, no toxicity was observed at 25 μM. At 50 μM, cellviability greater than 80% is entirely compatible with a genomicpharmacotoxicology study. The F2695 and F2696 compounds show nocytotoxicity in this assay, even at a concentration of 100 μM.

To perform the genomic pharmacotoxicological assessments, 3concentrations of the same compound are used: the concentration whichallows for 80% cell viability (C) to be obtained, as well asconcentrations corresponding to (C)×10 and to (C)/10.

In order to compare the capacity of F2695 and F2696 to yield a score inthe assay performed, the same concentrations were used in each test: 1μM, 10 μM and 100 μM. Concentrations of 1 μM, 10 μM and 50 μM were usedfor clomipramine. See FIGS. 5A, 5B and 5C.

4.3 Results on Primary Rat Hepatocytes

Toxicity Indices (TI) were determined as described above. Only thoseclones which were found to be altered in relation to the control weretaken into account in these indices, taking into consideration onlythose clones whose signal was two times higher than the backgroundthreshold (BT). Two separate analyses were performed using two levels ofdifferentiation (Induction Factor-IF) in relation to the untreatedcontrols:

-   -   a factor of at least 1.7 in relation to the untreated controls.        This factor of 1.7 times represents the weakest value that        allows an index not to be obtained in relation to the two        untreated controls.    -   a factor of at least 2 in relation to the untreated controls.        This factor of 2 times allows the most robust signals to be        taken into account.        4.3.1. Induction Factor of 1.7 in Relation to Untreated Control        (Table 7)

TABLE 7 Up- and down-regulated clones with primary rat hepatocytes(Induction Factor = 1.7 times) F2695- F2695- F2695- F2696- F2696- F2696-C 218- C 218- C 218- 1 μM 10 μM 100 μM 1 μM 10 μM 100 μM 1 μM 10 μM 100μM Up >1.7 Up 1 15 2 2 13 Down <0.588 Down 1 2 5 7 7 13 15 TI 2 2 5 22 915 28 Pos nb U nb D Gene A09 3 2.90 2.23 2.14 H. sapiens mitochondrion,12S A20 1 0.56 H. sapiens initiation factor elF-5A gene B20 2 0.14 0.27H. sapiens chromosome 19, BAC CIT-B-191n6 B22 2 0.17 0.32 H. sapiensGenomic sequence from 17 C01 4 3.20 1.93 1.82 1.91 H. sapiensmitochondrion, 16S E01 1 1.73 H. sapiens mRNA for lipocortin II E05 20.22 0.35 H. sapiens DNA sequence from clone 740A11 on chromosomeXq22.2-23. Contains part of the COL4A5 gene for Collagen Alpha 5 (IV)Chain Precursor. Contains GSS1, complete sequence E11 1 2.12 H. sapienschlordecone reductase homolog liver, mRNA E19 1 1.72 H. sapiensmitochondrion, cytochrome c oxidase subunit 1 E21 2 0.56 0.58 H. sapiensribosomal protein S14 gene F24 1 0.52 H. sapiens LIM homeobox proteincofactor (CLIM-1) mRNA G01 1 2.04 H. sapiens estrogen receptor- relatedprotein (variant ER from breast cancer) mRNA G05 1 2.02 H. sapiensmitochondrion, cytochrome c oxidase subunit 1 G09 2 2.09 1.76 H. sapiensmitochondrion, cytochrome b I01 1 2.05 H. sapiens mitochondrion,cytochrome c oxidase subunit 1 I18 2 2.38 1.88 H. sapiens 18S rRNA geneL0l 1 2.05 H. sapiens divalent cation tolerant protein CUTA mRNA L22 11.78 H. sapiens mRNA for Lon protease-like protein L23 1 1.75 H. sapienscDNA NIH_MGC_16 clone IMAGE: 3350241 5′, mRNA sequence M07 2 2.25 1.75H. sapiens mitochondrion, cytochrome c oxidase subunit 1 M12 3 0.21 0.160.39 H. sapiens mRNA; cDNA DKFZp564C1563 M23 1 1.95 Sequence 21 fromU.S. Pat. No. 5,851,764 P05 1 1.78 H. sapiens PAC clone DJ404K21 fromXq23 Q11 2 1.81 1.92 unk Q24 1 1.77 H. sapiens 28S ribosomal RNA geneS01 1 2.98 Mus muculus TCR beta locus T08 6 0.50 0.22 0.20 0.35 0.140.22 H. sapiens mRNA for KIAA1185 protein U04 6 0.57 0.26 0.19 0.48 0.220.37 H. sapiens translation initiation factor elF-2alpha mRNA V22 H.sapiens mRNA for elongation factor 1-alpha (clone CEF4) W17 1 2.96 H.sapiens mitochondrion, hypoxia inducible gene-14 X02 5 0.29 0.20 0.360.24 0.31 unk X05 2 0.15 0.24 H. sapiens microsomal epoxide hydrolase(EPHX) gene X06 5 0.2 0.16 0.23 0.15 0.23 H. sapiens Genomic sequencefrom 9q34 X23 1 1.92 unk Y17 1 2.65 H. sapiens 28S ribosomal RNA geneZ13 3 0.34 0.29 0.27 unk Z20 1 0.57 Homo sapiens cDNA wc44h09, x1NCI_CGAP_Pr28 clone IMAGE: 2321537 3′ similar to SW: RB24_Mouse P35290RAS_RELATED PROTEIN RAB-24;, mRNA sequence AA11 3 0.38 0.27 0.31 H.sapiens Repeat sequence AluJb fragment inserted into a cDNA coding foran unknown protein AA13 1 1.79 H. sapiens 18S rRNA gene AC13 5 0.22 0.160.28 0.16 0.28 H. sapiens 7S RNA L gene

The following Toxicity Indices were obtained:

F2695 Toxicity Index 1 μM 0 10 μM 0 100 μM 17 F2696 Toxicity Index 1 μM2 10 μM 5 100 μM 22 C218 Toxicity Index 1 μM 9 10 μM 15 50 μM 28

The following ranking could thus be established, from the most toxic tothe least toxic: C218 (clomipramine) >F2696>>>F2695.

Clomipramine, the reference molecule, coded C218 in the present study,showed an increasing number of signatures with relation to theconcentrations tested: respectively 9, 15 and 28 signatures atconcentrations of 1, 10 and 50 μM (maximal concentration defined in thepreliminary cytotoxicity test). As one might logically expect, all thesignatures that occurred at low and moderate concentrations are alsofound at the higher concentrations.

At concentrations of 1 and 10 μM, F2695 did not induce any of the 682potential signals of stress tested in the present study. At the highestconcentration, 100 μM, only two signatures were detected, one of whichwas common to both F2695 and C218, but whose signification was unknown.

F2696 showed an increasing number of signatures in relation to theconcentrations tested: 2, 5 and 22 signatures respectively atconcentrations of 1, 10 and 100 μM. All of the signatures that occurredat the low and medium concentrations were detected at the higherconcentrations. None of the 22 signatures was shared with F2695.Conversely, all 5 of the signatures that appeared at the low and mediumconcentration (5 including the 2 which were present at the lowconcentration) were among the 9 signatures detected with clomipraminestarting with the low dose, 1 μM. At the high concentration, 100 μM, 10of the 26 signatures of F2696 were detected among the 28 signaturesidentified with clomipramine at 50 μM.

From a qualitative standpoint, the impact of F2696 and of clomipramineon mitochondrial transcripts, in particular on Cox1 and on cytochrome b,should be stressed. These signatures are not present with F2695(G05/G09/I01 positions).

4.3.2. Induction Factor of 2 in Relation to Untreated Control (Table 8)

TABLE 8 Up- and down-regulated clones with primary rat hepatocytes(Induction Factor = 2 times) F2695- F2695- F2695- F2696- F2696- F2696- C218- C 218- C 218- 1 μM 10 μM 100 μM 1 μM 10 μM 100 μM 1 μM 10 μM 100 μMUp >1.7 Up 10 1 1 4 Down <0.588 Down 5 6 7 12 12 TI 5 16 8 13 16 Pos nbU nb D Gene A09 3 2.90 2.23 2.14 H. sapiens mitochondrion, 12S B20 20.14 0.27 H. sapiens chromosome 19, BAC CIT-B-191n6 B22 2 0.17 0.32 H.sapiens Genomic sequence from 17 C01 1 3.20 H. sapiens mitochondrion,16S E05 2 0.22 0.35 H. sapiens DNA sequence from clone 740A11 onchromosome Xq22.2-23. Contains part of the COL4A5 gene for CollagenAlpha 5 (IV) Chain Precursor. Contains GSS1, complete sequence E11 12.12 H. sapiens chlordecone reductase homolog liver, mRNA G01 1 2.04 H.sapiens estrogen receptor- related protein (variant ER from breastcancer) mRNA G05 1 2.02 H. sapiens mitochondrion, cytochrome c oxidasesubunit 1 G09 1 2.09 H. sapiens mitochondrion, cytochrome b I01 1 2.05H. sapiens mitochondrion, cytochrome c oxidase subunit 1 I18 1 2.38 H.sapiens 18S rRNA gene J03 1 2.12 H. sapiens CLP mRNA L01 1 2.05 H.sapiens divalent cation tolerant protein CUTA mRNA M07 1 2.25 H. sapiensmitochondrion, cytochrome c oxidase subunit 1 M12 3 0.21 0.16 0.39 H.sapiens mRNA; cDNA DKFZp564C1563 S01 1 2.98 Mus muculus TCR beta locusT08 5 0.22 0.20 0.35 0.14 0.22 H. sapiens mRNA for KIAA1185 protein U045 0.26 0.19 0.48 0.22 0.37 H. sapiens translation initiation factorelF-2alpha mRNA W17 1 2.96 H. sapiens mitochondrion, hypoxia induciblegene-14 X02 5 0.29 0.20 0.36 0.24 0.31 unk X05 2 0.15 0.24 H. sapiensmicrosomal epoxide hydrolase (EPHX) gene X06 5 0.20 0.16 0.23 0.15 0.23H. sapiens Genomic sequence from 9q34 Y17 1 2.65 H. sapiens 28Sribosomal RNA gene Z13 3 0.34 0.29 0.27 unk AA11 3 0.38 0.27 0.31 H.sapiens Repeat sequence AluJb fragment inserted into a cDNA coding foran unknown protein AC13 5 0.22 0.16 0.28 0.16 0.28 H. sapiens 7S RNAgene

The following Toxicity Indices were obtained:

F2695 Toxicity Index 1 μM 0 10 μM 0 100 μM 0 C218 Toxicity Index 1 μM 810 μM 13 50 μM 16 F2696 Toxicity Index 1 μM 0 10 μM 5 100 μM 16

According to theses parameters, the following ranking could be putforward, from the most toxic to the least toxic: C218(clomipramine)>F2696>>>>>F2695.

With regard to over- and under-expressed clones at a Factor of 2, F2695did not induce any signatures, even at a concentration of 100 μM.

The concentration effect on the occurrence of signatures was confirmedby the fact that the weak signatures with F2696 at 1 μM, which werepresent in the preceding analysis with the Factor of 1.7, disappear.

From a qualitative standpoint, the impact of F2696 and of clomipramineon Cox1 and on cytochrome b was also confirmed (G05/G09/I01 positions).

F2695, the pharmacologically-active enantiomer of F2207, was withoutsignificant impact in this test, whereas clomipramine is used aspositive-control reference product.

Conversely, F2696, the pharmacologically-inactive enantiomer of F2207,showed a profile of signatures that is quantitatively and qualitativelyclose to that of clomipramine, and shows no signatures in common withF2695.

All of this is evidence of a superior toxico-genomic profile for theactive F2695 enantiomer which, in this experimental model, had a verysignificantly better safety coefficient that that of F2696.

4.4 Conclusion

The genomic pharmacotoxicology studies performed on the F2695 and F2696molecules, enantiomers of F2207 (at concentrations of 10, 50 and 100μM), and on C218 (clomipramine, at concentrations of 1, 10 and 50 μM),using rat hepatocytes in primary culture, yieldedconcentration-dependent stress signatures and Toxicity Indices. Thesestudies confirm the capacity of the genomic pharmacotoxicology test toreveal stress signatures under treatment conditions (concentrations,duration of treatment) that do not cause any toxicity in a classiccell-viability assay such as MTT-assay.

This study brings to light several important facts:

-   -   in the primary rat hepatocyte model, only F2695, the        pharmacologically-active enantiomer of F2207, did not induce a        significant Toxicity Index;    -   F2696, the pharmacologically-inactive enantiomer of F2207, and        clomipramine, the reference psychotropic product, induced marked        Indices involving very similar or common stress signatures. In        this system, clomipramine, the positive-control reference        product, induced the highest number of stress signatures,        significant indices having been observed starting at the lowest        concentrations. On this subject, it is interesting to note that        clomipramine can induce a certain number of adverse events in        man, such as tachycardia, orthostatic hypotension, cardiac        conduction or rhythm disturbances, and exceptionally hepatitis.        In case of accidental overdosage with clomipramine, syncope,        haematological disturbances and severe cardiovascular        manifestations can be observed.

Without inferring a common physiopathological mechanism, it isinteresting to note that F2696 showed very similar or common stresssignatures to those of clomipramine and also induces adverse events suchas the cardiovascular disturbances previously described.

Thus, it is legitimate to suggest that the signatures observed areindependent of any antidepressant, or more broadly psychotropic,profile. On the contrary, the signatures should indeed be considered tobe “signatures of stress” (F2696 causes in particular reduced expressionof a gene involved in protein synthesis and of a translation initiationfactor). All of this is evidence of a superior toxico-genomic profilefor the active F2695 enantiomer which, in this experimental model, had avery significantly better safety coefficient that that of F2696.

The invention claimed is:
 1. A method for treating a patient afflictedwith a condition or disorder which may be treated by double inhibitionof serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting therisks of cardiovascular disturbances and/or the risks of organ and/ortissue toxicity, comprising the step of administering to the patient anamount of a mixture of enantiomers of milnacipran hydrochloride(Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamidehydrochloride), such mixture being substantially pure in the dextrogyralenantiomer, effective for alleviation of the condition or disorder,wherein the administration of said mixture limits the risks ofcardiovascular disturbances and/or the risks of organ and/or tissuetoxicity, relative to administration of racemic milnacipranhydrochloride.
 2. The method of claim 1, wherein the cardiovasculardisturbance corresponds to an increase in blood pressure and/or anincrease in heart rate.
 3. The method of claim 2, wherein the increasein blood pressure corresponds to an increase in diastolic bloodpressure.
 4. The method of claim 1, wherein the organ toxicity iscardiac toxicity and the tissue toxicity is hepatic and/or renaltoxicity.
 5. The method of claim 1, wherein mass/mass ratio between thedextrogyral enantiomer and levogyral enantiomer in the mixture isgreater than 95:5 (dextrogyral:levogyral).
 6. The method of claim 1,wherein mass/mass ratio between the dextrogyral enantiomer and levogyralenantiomer in the mixture is greater than 99:1 (dextrogyral:levogyral).7. The method of claim 1, wherein mass/mass ratio between thedextrogyral enantiomer and levogyral enantiomer in the mixture isgreater than 99:5:0.5 (dextrogyral:levogyral).
 8. The method of claim 1,wherein the disorder or condition is selected from depression, bi-polardisease, schizophrenia, generalised anxiety, morose and marasmic states,stress-related diseases, panic attacks, phobias, obsessive-compulsivedisorders, behavioural disorders, oppositional disorders, post-traumaticstress disorder, depression of the immune System, fatigue and theassociated pain syndromes, chronic fatigue syndrome, fibromyalgia, andother functional somatic disorders, autism, disorders characterised byattention deficit due to general health status, attention disorders dueto hyperactivity, eating disorders, neurotic bulimia, neurotic anorexia,obesity, psychotic disorders, apathy, migraine, pain, irritable bowelsyndrome, cardiovascular diseases, neuro-degenerative diseases and theassociated anxiety-depressive syndromes (Alzheimer's disease,Huntington's chorea, Parkinson's disease), urinary incontinence and drugaddiction.
 9. The method of claim 8, wherein depression is selected fromdeep depression, resistant depression, depression in the elderly,psychotic depression, depression induced by treatments with interferon,depressive state, manic-depressive syndrome, seasonal depressiveepisodes, depressive episodes related to general health status,depression related to mood-altering substances.
 10. The method of claim8, wherein the phobia is agoraphobia.
 11. The method of claim 8, whereinthe pain is chronic pain.
 12. The method of claim 8, wherein thecardiovascular disease is selected from anxiety-depressive syndrome inmyocardial infarct or in hypertension.
 13. The method of claim 8,wherein the urinary incontinence is selected from urinary incontinencerelated to stress and enuresis.
 14. The method of claim 8, wherein thedrug addiction is selected from anxiety addiction to tobacco, tonicotine, to alcohol, to narcotics, to drugs, and to an analgesic usedin weaning-off from these addictive states.
 15. The method of claim 1,wherein the patient is selected from children, the elderly, patientswith hepatic and/or renal insufficiency, patients receiving treatmentthat induces hepatic or renal organ and/or tissue toxicity, patientsreceiving treatment for a heart condition, patients receiving treatmentthat induces cardiovascular side-effects, patients having a history ofcardiovascular disease and/or suffering from cardiovascular disorders.16. The method of claim 15, wherein the history of cardiovasculardisease and/or cardiovascular disorders are chosen among myocardialinfarct, cardiac rhythm disorders (tachycardia, bradycardia,palpitations), blood pressure disorders (hypo- or hypertensive patients)and heart disease.
 17. A method for treating a patient afflicted withdepression, while limiting the risks of cardiovascular disturbancesand/or the risks of organ and/or tissue toxicity, comprising the step ofadministering to the patient an amount of: a) a mixture of enantiomerssubstantially pure in the dextrogyral enantiomer of milnacipranhydrochloride(Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamidehydrochloride), wherein the administration of said mixture limits therisks of cardiovascular disturbances and/or the risks of organ and/ortissue toxicity, relative to administration of racemic milnacipranhydrochloride, and b) at least one active compound selected from thepsychotropics, as associated products for use simultaneously, separatelyor staggered in time, effective for alleviation of depression.
 18. Themethod of claim 17, wherein the psychotropics are selected fromantidepressants and antimuscarinic agents.
 19. The method according toclaim 17, wherein the depression is selected from deep depression,resistant depression, depression in the elderly, psychotic depression,depression induced by treatments with interferon, depressive state,manic-depressive syndrome, seasonal depressive episodes, depressiveepisodes related to general health status, depression related tomood-altering substances.
 20. A method for treating a patient afflictedwith a condition or disorder which may be treated by double inhibitionof serotonin (5-HT) and norepinephrine (NE) reuptake, while limiting therisks of cardiovascular disturbances and/or the risks of organ and/ortissue toxicity, comprising the step of administering to the patient anamount of: a) a mixture of enantiomers substantially pure in thedextrogyral enantiomer of milnacipran hydrochloride(Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamidehydrochloride), wherein the administration of said mixture limits therisks of cardiovascular disturbances and/or the risks of organ and/ortissue toxicity, relative to administration of racemic milnacipranhydrochloride, and b) at least one other active substance selected fromthe active compounds that induce organ toxicity and the active compoundsthat induce cell toxicity, as associated products for usesimultaneously, separately or staggered in time, effective foralleviation of the condition or disorder.
 21. The method of claim 20,wherein the cell toxicity is hepatic and/or renal toxicity.
 22. A methodfor treating a patient afflicted with a condition or disorder which maybe treated, by double inhibition of serotonin (5-HT) and norepinephrine(NE) reuptake while limiting the risks of cardiovascular disturbancesand/or the risks of organ and/or tissue toxicity, comprising the step ofadministering to the patient an amount of: a) a mixture of enantiomerssubstantially pure in the dextrogyral enantiomer of milnacipranhydrochloride(Z(±)-2-(aminomethyl)-N,N-diethyl-1-phenylcyclopropanecarboxamidehydrochloride), wherein the administration of said mixture limits therisks of cardiovascular disturbances and/or the risks of organ and/ortissue toxicity, relative to administration of racemic milnacipranhydrochloride, and b) at least one other active substance selected fromthe active compounds that induce cardiovascular side-effects, asassociated products for use simultaneously, separately or staggered intime, effective for alleviation of the condition or disorder.
 23. Themethod of claim 8, wherein the disorder or condition is depression andwherein the mixture of enantiomers is administered as a sustainedrelease pharmaceutical form having a dose ranging from 0.01 mg/kg to 10mg/kg body weight per day in one or more intakes.
 24. The method ofclaim 23, wherein the sustained release pharmaceutical form has a doseranging from 0.05 mg/kg to 5 mg/kg body weight per day in one or moreintakes.
 25. The method of claim 24, wherein the sustained releasepharmaceutical form has a dose ranging from 0.1 mg/kg to 1 mg/kg bodyweight per day in one or more intakes.